Immunotherapy of b cell malignancies and autoimmune diseases using unconjugated antibodies and conjugated antibodies and antibody combinations and fusion proteins

ABSTRACT

The invention is directed to a method for treating a treating and diagnosing a B cell-related disease, T cell-related disease or an autoimmune disease in a mammal by concurrently or sequentially administering to the mammal a therapeutic composition that comprises a pharmaceutically acceptable vehicle and at least one conjugated antibody, wherein predosing with a non-radiolabeled antibody is not performed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on U.S. provisional patent application Ser.No. 60/437,145, filed Dec. 31, 2002. The entire contents of thisapplication, including its specification, claims and drawings, areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to immunotherapeutic method for treatingB-cell related malignancies, particularly aggressive non-Hodgkin'slymphomas. In particular, this invention is directed to methods fortreating and diagnosing a B cell-related disease, T cell-related diseaseor an autoimmune disease in a mammal by administering to the mammal atherapeutic composition, wherein predosing with a non-radiolabeledantibody is not performed.

BACKGROUND OF THE INVENTION

B-cell lymphomas express surface antigens that have shown to be goodtargets for therapy with monoclonal antibodies (Mab). Antibodies, eitherused alone (naked antibodies) or in conjunction with chemotherapy, canbe conjugated with toxins or with radionuclides for radioimmunotherapy(RAIT). The radiolabelled antibody is administered after (Kaminski, M.S. et al., J. Clin. Oncol. 19:3918-3928, 2001) or together (Press, O. W.et al, New Engl. J. Med. 329:1219-24, 1993) with unlabelled antibody toimprove dose distribution. Most investigators use a radiolabeled mouseantibody combined with an unlabeled antibody, which is murine orchimeric. It has been considered advantageous to radiolabel a mouseantibody from a toxicological point of view due to its shorter half-lifecompared to a chimeric antibody. A Mab with longer half-life gives alonger residence time of the radioimmunoconjugate in blood and bonemarrow and probably thus induces more toxicity. Since the antibody inits own right hardly induces toxicity both mouse and chimeric unlabelledantibodies are used to improve dose distribution by allegedly saturatingantigen on normal cells and tissues in the body (cf. Kaminski, U.S. Pat.No. 5,595,721; Wiseman et al., Crit. Rev. Oncol. Hematol. 39:181-194,2001).

The use of monoclonal antibodies in targeted radiotherapy of cancers(radioimmunotherapy; RAIT) has produced striking clinical responses inhematologic diseases such as non-Hodgkin's lymphoma (NHL). Newstrategies are presently examined in an effort to minimize the systemictoxicity of a circulating radionuclide and the sensitization of tumorsby radiation. The former being carried out by pretargeting and thelatter by combination therapy with radiosenzitizing drugs. See Govindan,S. V et al., Current Trends, Pharmaceutical Science and Technology Today3:90-98, 2000.

The anti-tumor activity of RAIT is mainly due to the associatedradioactivity of the radiolabel attached to the antibody, which emitscontinuous, exponentially decreasing low-dose-rate irradiation with aheterogeneous dose deposition. Four radiolabeled antibody products areprogressing towards commercialization for the RAIT of NHL. They include¹³¹I-tositumomab (Bexxar™), ⁹⁰Y-ibritumomab tiuxetan (Zevalin™),⁹⁰Y-epratuzumab (hLL2) and ¹³¹I-Lym-1. For a more detail review of theseproducts, see Goldenberg, D. M., Critical Reviews in Oncology/Hematology39:195-201, 2001, and Goldenberg, D. M., J. Nucl. Med. 43:693-713, 2002.

Bexxar (Corixa Corp., Seattle, Wash.) and Zevalin (IDEC-Y2B8; IDECPharmaceuticals, San Diego, Calif.) are both murine monoclonalantibodies (Mabs) directed against CD20 antigen expressed in the surfaceof normal and malignant B-lymphocytes. Bexxar is used as an IgG2a murineMab with cold murine antibody added, whereas Zevalin has the murineantibody labeled and cold human.mouse chimeric rituximab (Rituxan™,IDEC-Genentech) added to the product. Both products provide forpretherapy cold antibody dosing in order to improve tumor targeting,which involves a 1-h infusion of 450 mg of unlabeled Bexxar antibody anda 4-6 h infusion of 450 mg of rituximab with Zevalin. Both products haveshown a higher and more durable responses than naked antibodies,however, they also have dose-limiting toxicity, predominantlymyelotoxicity. Zevalin was approved by the Food and Drug Administration(FDA) for the treatment of recurrent low grade or transformed B cellnon-Hodgkin's lymphoma. These radiolabeled anti-CD-20 Mab must bepreceded by a dose of cold antibodies to enable good tumor localization.In fact, the specific localization numbers for mindium -Zevalin dropfrom 78% to 15% tumor uptake at specific tumor sites when predosing isinvolved (Wiseman et al., ibid).

Epratuzumab (⁹⁰Y -epratuzumab) is a humanized IgG₁ antibody directedagainst the anti-CD22 antigen. The antigen is fast internalized uponantibody binding. The naked antibody has been reported to show efficacyin follicular as well as diffuse large B-cell lymphoma (Leonard, J. P.et al., Epratuzumab (hLL2, anti-CD22 humanized monoclonal antibody) isan active and well-tolerated therapy for refractory/relapsed diffuselarge B-cell non-Hodgkin's lymphoma (NHL). Blood (Suppl) 96:578a [abstr.2482], 2000; Press, O. W. et al., Immunotherapy of Non-Hodgkin'sLymphomas. Hematology (Am. Soc. Hematol. Educ. Program), p. 221-40,2001). Epratuzumab is not expected to give rise to human anti-humanantibodies (HAHA), which makes it suited for repeated dosing. The mouseparental antibody, mLL2,labelled with ¹³¹I and has shown efficacy invarious subtypes of B-cell lymphoma (Linden, O. et al. Clin. Cancer Res.5:3287s-3291s, 1999). After internalization, the ¹³¹I-labelled antibodyis dehalogenated and the radionuclide is released from the cell.Radiometals like yttrium are retained in the cell upon internalization(Sharkey, R. M., et al. Cancer Immunol. Immunother. 44:179-88, 1997).The shorter physical half-life of ⁹⁰Y compensates in some degree for thelonger half-life of epratuzumab and provides the rational for theircombination.

RAIT is usually given as a single infusion. There are, however,theoretical advantages of a fractionated approach, since fractionationwould better deal with the problem of heterogeneity in absorbed dose, asoutlined in O'Donoghue, J. A., Dosimetric Principles of TargetedRadiotherapy, in Radioimmunotherapy of Cancer, A. R. Fritzberg (ed.),Marcel Dekker, Inc., p. 1-20, New York, Basel, 2000. There are alsoexperimental data supporting that therapeutic response can be improvedby splitting a large single administration of radiolabelled antibodyinto a number of smaller administrations (Schlom, J. et al. J. Natl.Cancer Inst. 82:763-71, 1990). Approaches with two infusions as well asmultiple have been explored clinically using mouse antibodies (DeNardo,G. L., et al. Cancer Biother. Radiopharm. 13:239-54, 1998; Vose, J. M.,et al . J Clin. Oncol. 18:1316-23, 2000).

Intratumoral variability in the expression of CD22 antigen has beenreported. In fresh tumor samples from five patients, 52-89% of lymphomacells were found to bear the antigen for the anti-CD22 MAb HD6 (Press,O. W. et al. Cancer Res. 49:4906-12, 1989). One alleged advantage ofRAIT using long range β-emitters is their ability to kill antigennegative tumour cells in the vicinity of the targeted cells. Byassessing the antigen expression of tumour cells before therapy, onecould study the clinical relevance of this concept in the setting ofRAIT using the anti-CD22 ⁹⁰Y-labelled epratuzumab.

Research was undertaken to confirm the theoretical advantages of dosefractionation and the published experimental data that support it. Thestudy was intended to investigate the feasibility of fractionated RAIT,using a radiolabeled humanized antibody. It was found that afterpredosing with 100 mg of the humanized CD22 Mab, epratuzumab, labelledwith ¹¹¹In for dosimetry purposes, subsequent fractionated doses of⁹⁰Y-labelled epratuzumab at doses of up to 7.5 mCi/m², once weekly forup to 2-3 weeks, resulted in tolerable and effective radioimmunotherapy(Linden et al., Cancer Biother Radiopharm 2002; 17: 490 [abstract 47].Although these clinical studies suggest that fractionated therapy of aradioimmunoconjugate is feasible, no comparison was made withadministered a single high-dose of the radioimmunoconjugate in terms ofsafety and efficacy. Since the first “dosimetry” dose with ¹¹¹Incontained 100 mg of antibody, and each susccessive injection alsocontained this naked antibody dose, it also could not be determined ifthese doses that totalled at least 300 mg of epratuzumab also served asa predosing effect as suggested in other cited studies involving CD20antibodies. Therefore, it was not interpretable from these studieswhether or not any predosing was needed for such radioimmunotherapy,particularly with CD22 antibodies.

We have now found that predosing is not used in this invention, contraryto other published studies and Kaminski's U.S. Pat. No. 5,595,721, tosaturate the antigenic sites in the normal tissues and spleen, aspracticed in the prior art. Clearly, the invention disclosed hereinshows that there is a lack of a need of high antibody predosing, aspracticed in the prior art.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide methodsfor treating a disease in a mammal by administering a therapeuticcomposition wherein predosing with a non-radiolabeled antibody, fragmentor fusion protein is not performed.

It is also an object of the current invention to make theabove-mentioned methods not only simple and easy for administration, yetby themselves, remain therapeutically active and have similar responserates without having a higher dose of naked antibody affecting thetumor.

It is further an object of the invention to provide methods that show amore effective response in treating aggressive non-Hodgkin's lymphoma,in contrast to what is demonstrated by the prior art that only showseffects in indolent forms of lymphoma.

These and other objects are achieved, in accordance with an embodimentof the present invention, by provision of a method for treating adisease in a mammal comprising concurrently or sequentiallyadministering to the mammal a therapeutic composition that comprises apharmaceutically acceptable vehicle and at least one conjugated antibodyor a fragment thereof or a conjugated antibody fusion protein or afragment thereof, wherein predosing with a non-radiolabeled antibody,fragment or fusion protein is not performed. The unconjugated antibody,fragment or fusion protein is optionally added with the conjugatedantibody, fragment or fusion protein, as a maintenance therapy to keeptumor cells from target escape.

In a preferred embodiment, the present invention is directed to a methodfor treating diseases such as B-cell-related malignancies. In addition,it is also useful for treating autoimmune diseases, as well asT-cell-related malignancies.

In another preferred embodiment, the conjugated and unconjugatedantibodies, fragments, and fusion proteins of the present invention canbe targeted against an antigen selected from the group consisting ofCD3, CD4, CD5, CD8, CD11c, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD52, CD54, CD74, CD80, CD126, Ia,HMI.24, HLA-DR, tenascin, MUC1 and B-cell-tumor-associated antigens,including vascular endothelial antigens, such as vascular endothelialgrowth factor (VEGF) and placenta growth factor (P1GF). In a relatedvein, the conjugated and/or unconjugated antibodies, fragments or fusionproteins of the present invention can be the same or different. Inaddition, these antibodies can be human, murine, chimeric, subhumanprimatized or humanized. Furthermore, these antibodies, fragments orfusion proteins can be selected from the group consisting of intact IgG,F(ab′)₂, F(ab)₂, Fab′, Fab, scFvs, diabodies, triabodies or tetrabodiesand can be conjugated to at least one therapeutic agent.

In accordance with another aspect of the present invention, a method isprovided as described above, wherein mammalian subjects, such as humansand domestic or companion animals, are treated with one or moreantibodies that are conjugated to one or more therapeutic agentsselected from the group consisting of drug, toxin, immunomodulator,chelator, boron compounds, photodynamic agent, and radionuclide.

In yet another preferred embodiment, the therapeutic compositioncomprises a fusion protein of said combination of antibodies orantibodies with immunomodulators. The fused antibodies can compriseantibodies against different antigens as well as antibodies againstdifferent epitopes of the same antigen.

The present invention contemplates the above-mentioned method whereinthe conjugated and unconjugated antibody is an anti-CD22 monoclonal thatis parenterally administered into a mammal at a preferable dosage of20-600 milligrams protein per dose, more preferably at 20-150 milligramsprotein per dose, and most preferably, at 20-100 milligrams protein perdose. In addition, the mammal may receive the anti-CD22 antibody asrepeated parenteral dosages of preferably 20-150 milligrams protein perdose and more preferably, 20-100 mg protein per dose. It is important torecognize that such doses are given as the actual therapeutic dosewithout requiring any predosing, either for improving targeting or fordosimetric purposes, as practiced previously by, for example, Juweid etal., Clin. Cancer Res. 5:3292s-3303s, 1999 (where a prior dose of 50 mgof the CD22 Mab conjugated with ¹¹¹In or another diagnostic isotope wasrequired). No attempt was made in such studies to assess the ability ofthe therapeutic radioimmunoconjugate with various protein doses of theantibody to be effective directly without a prior dosing regimen.

In another preferred embodiment, the method for treating a disease in amammal comprises administering to the mammal a therapeutic compositioncomprising a pharmaceutically acceptable vehicle and a multispecificmultivalent antibody, fragment or fusion protein conjugate that binds toat least one target antigen and a therapeutic agent, wherein predosingwith a non-radiolabeled antibody is not performed.

In yet another preferred embodiment, the method for treating a diseasein mammals comprises:

(a) administering to the mammal a composition that comprises amultispecific multivalent antibody, fragment or fusion protein thatbinds to at least one target antigen;

(b) optionally, a clearing agent to allow the composition to clearnon-localized antibodies from circulation; and

(c) administering to the mammal a pharmaceutially effective amount oftherapeutic conjugate that binds to the multispecific multivalentantibody, fragment or fusion protein,

and wherein predosing with a non-radiolabeled antibody is not performed.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description and appendedclaims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise specified, “a” or “an” means “one or more”.

1. Definitions

In the description that follows, a number of terms are used and thefollowing definitions are provided to facilitate understanding of thepresent invention.

Non-Hodgkin's lymphoma (NHL) refers to a family of lymphoma diseasesthat involves lymph nodes, spleen, other organs and often the bonemarrow There are at least 30 different types of NHL. The two commontypes are follicular (low grade or indolent) and aggressive, diffuselarge cell (intermediate or high grade) lymphomas.

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. For example, an anti-CD22 monoclonal antibody fragmentbinds with an epitope of CD22. The term “antibody fragment” alsoincludes any synthetic or genetically engineered protein that acts likean antibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), and minimal recognitionunits consisting of the amino acid residues that mimic the hypervariableregion.

A naked or cold antibody is generally an entire antibody which is notconjugated (unconjugated) to a therapeutic agent. This is so because theFc portion of the antibody molecule provides effector functions, such ascomplement fixation and ADCC (antibody dependent cell cytotoxicity),which set mechanisms into action that may result in cell lysis. However,it is possible that the Fc portion is not required for therapeuticfunction, with other mechanisms, such as apoptosis, coming into play.Naked antibodies are also non-radiolabeled antibodies that include bothpolyclonal and monoclonal antibodies, as well as certain recombinantantibodies, such as primatized subhuman, chimeric, humanized or humanantibodies.

A chimeric antibody is a recombinant protein that contains the variabledomains including the complementarity determining regions (CDRs) of anantibody derived from one species, preferably a rodent antibody, whilethe constant domains of the antibody molecule is derived from those of ahuman antibody. For veterinary applications, the constant domains of thechimeric antibody may be derived from that of other species, such as acat or dog.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species; e.g., a rodent antibody, is transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains. The constant domains of the antibodymolecule is derived from those of a human antibody.

A human antibody is an antibody obtained from transgenic mice that havebeen “engineered” to produce specific human antibodies in response toantigenic challenge. In this technique, elements of the human heavy andlight chain locus are introduced into strains of mice derived fromembryonic stem cell lines that contain targeted disruptions of theendogenous heavy chain and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Greenet al., Nature Genet. 7:13 (1994), Lonberg et al., Nature 368:856(1994), and Taylor et al., Int. Immun. 6:579 (1994). A fully humanantibody also can be constructed by genetic or chromosomal transfectionmethods, as well as phage display technology, all of which are known inthe art. See for example, McCafferty et al., Nature 348:552-553 (1990)for the production of human antibodies and fragments thereof in vitro,from immunoglobulin variable domain gene repertoires from unimmunizeddonors. In this technique, antibody variable domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, and displayed as functional antibody fragments on thesurface of the phage particle. Because the filamentous particle containsa single-stranded DNA copy of the phage genome, selections based on thefunctional properties of the antibody also result in selection of thegene encoding the antibody exhibiting those properties. In this way, thephage mimics some of the properties of the B cell. Phage display can beperformed in a variety of formats, for their review, see e.g. Johnsonand Chiswell, Current Opiniion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, which are incorporated intheir entirety by reference.

A therapeutic agent is a molecule or atom which is administeredseparately, concurrently or sequentially with an antibody moiety orconjugated to an antibody moiety, i.e., antibody or antibody fragment,or a subfragment, and is useful in the treatment of a disease. Examplesof therapeutic agents include antibodies, antibody fragments, drugs,toxins, nucleases, hormones, immunomodulators, chelators, boroncompounds, photoactive agents or dyes and radioisotopes.

An immunomodulator is a therapeutic agent as defined in the presentinvention that when present, alters, suppresses or stimulates the body'simmune system. Typically, the immunomodulator useful in the presentinvention stimulates immune cells to proliferate or become activated inan immune response cascade, such as macrophages, B-cells, and/orT-cells.

An immunoconjugate is a conjugate of an antibody component with atherapeutic or diagnostic agent. The diagnostic agent can comprise aradioactive or non-radioactive label, a contrast agent (such as formagnetic resonance imaging, computed tomography or ultrasound), and theradioactive label can be a gamma-, beta-, alpha-, Auger electron-, orpositron-emitting isotope.

An expression vector is a DNA molecules comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells, as well as an transgenicanimal, that have been genetically engineered to contain the clonedgene(s) in the chromosome or genome of the host cell or cells of thehost cells. Suitable mammalian host cells include myeloma cells, such asSP2/0 cells, and NSO cells, as well as Chinese Hamster Ovary (CHO)cells, hybridoma cell lines and other mammalian host cell useful forexpressing antibodies. Also particularly useful to express mAbs andother fusion proteins, is a human cell line, PER.C6 disclosed in WO0063403 A2, which produces 2 to 200-fold more recombinant protein ascompared to conventional mammalian cell lines, such as CHO, COS, Vero,Hela, BHK and SP2-cell lines. Special transgenic animals with a modifiedimmune system are particularly useful for making fully human antibodies.

As used herein, the term antibody fusion protein is a recombinantlyproduced antigen-binding molecule in which two or more of the same ordifferent single-chain antibody or antibody fragment segments with thesame or different specificities are linked. Valency of the fusionprotein indicates how many binding arms or sites the fusion protein hasto a single antigen or epitope; i.e., monovalent, bivalent, trivalent ormutlivalent. The multivalency of the antibody fusion protein means thatit can take advantage of multiple interactions in binding to an antigen,thus increasing the avidity of binding to the antigen. Specificityindicates how many antigens or epitopes an antibody fusion protein isable to bind; i.e., monospecific, bispecific, trispecific,multispecific. Using these definitions, a natural antibody, e.g., anIgG, is bivalent because it has two binding arms but is monospecificbecause it binds to one epitope. Monospecific, multivalent fusionproteins have more than one binding site for an epitope but only bindswith one epitope, for example a diabody with two binding site reactivewith the same antigen. The fusion protein may comprise a single antibodycomponent, a multivalent or multispecific combination of differentantibody components or multiple copies of the same antibody component.The fusion protein may additionally comprise an antibody or an antibodyfragment and a therapeutic agent. Examples of therapeutic agentssuitable for such fusion proteins include immunomodulators(“antibody-immunomodulator fusion protein”) and toxins (“antibody-toxinfusion protein”). One preferred toxin comprises a ribonuclease (RNase),preferably a recombinant RNase.

A multispecific antibody is an antibody that can bind simultaneously toat least two targets that are of different structure, e.g., twodifferent antigens, two different epitopes on the same antigen, or ahapten and/or an antigen or epitope. One specificity would be for aB-cell, T-cell, myeloid-, plasma-, and mast-cell antigen or epitope.Another specificity could be to a different antigen on the same celltype, such as CD3, CD4, CD5, CD8, CD11c, CD14, CD15, CD19, CD20, CD21,CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD52, CD54, CD74, CD80,CD126, Ia, HMI.24, HLA-DR, tenascin, MUC1 and a B-cell-tumor-associatedantigen, including vascular endothelial antigens, such as VEGF and P1GF.Multispecific, multivalent antibodies are constructs that have more thanone binding site, and the binding sites are of different specificity.For example, a diabody, where one binding site reacts with one antigenand the other with the another antigen.

A bispecific antibody is an antibody that can bind simultaneously to twotargets which are of different structure. Bispecific antibodies (bsAb)and bispecific antibody fragments (bsFab) have at least one arm thatspecifically binds to, for example, a B-cell, T-cell, myeloid-, plasma-,and mast-cell antigen or epitope and at least one other arm thatspecifically binds to a targetable conjugate that bears a therapeutic ordiagnostic agent. A variety of bispecific fusion proteins can beproduced using molecular engineering. In one form, the bispecific fusionprotein is monovalent, consisting of, for example, a scFv with a singlebinding site for one antigen and a Fab fragment with a single bindingsite for a second antigen. In another form, the bispecific fusionprotein is divalent, consisting of, for example, an IgG with a bindingsite for one antigen and two scFv with two binding sites for a secondantigen.

Caninized or felinized antibodies are recombinant proteins in whichrodent (or another species) complementarity-determining regions of amonoclonal antibody have been transferred from heavy and light variablechains of rodent (or another species) immunoglobulin into a dog or cat,respectively, immunoglobulin variable domain.

Subhuman primatized antibodies are recombinant proteins in whichsubhuman primate (e.g., monkey) complementarity-determining regions of amonoclonal antibody have been transferred from heavy and light varianchains of roden (or another species) immunoglobulin into a subhumanprimate immunoglobulin variable domain.

Domestic animals include large animals such as horses, cattle, sheep,goats, llamas, alpacas, and pigs, as well as companion animals. In apreferred embodiment, the domestic animal is a horse.

Companion animals include animals kept as pets. These are primarily dogsand cats, although small rodents, such as guinea pigs, hamsters, rats,and ferrets, are also included, as are subhuman primates such asmonkeys. In a preferred embodiment the companion animal is a dog or acat.

The term “clearing agent” refers to an antibody which binds the bindingsite of the targeting moiety, wherein the targeting moiety can be anantibody, an antigen-binding antibody fragment or a non-antibodytargeting moiety. In a more preferred method, the clearing agent is amonoclonal antibody that is an anti-idiotypic to the monoclonal antibodyof the conjugate used in the first step, as described in U.S.application Ser. No. 08/486,166. In another preferred embodiment, theclearing agent is substituted with multiple residues of carbohydrate,such as galactose, which allow the clearing agent to be cleared quicklyfrom circulation by asialoglycoprotein receptors in the liver.

2. Preparation of Monoclonal Antibodies including Chimeric, Humanizedand Human Antibodies

Monoclonal antibodies (MAbs) are a homogeneous population of antibodiesto a particular antigen and the antibody comprises only one type ofantigen binding site and binds to only one epitope on an antigenicdeterminant.

Rodent monoclonal antibodies to specific antigens may be obtained bymethods known to those skilled in the art. See, for example, Kohler andMilstein, Nature 256: 495 (1975), and Coligan et al. (eds.), CURRENTPROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons1991) [hereinafter “Coligan”]. Briefly, monoclonal antibodies can beobtained by injecting mice with a composition comprising an antigen,verifying the presence of antibody production by removing a serumsample, removing the spleen to obtain B-lymphocytes, fusing theB-lymphocytes with myeloma cells to produce hybridomas, cloning thehybridomas, selecting positive clones which produce antibodies to theantigen, culturing the clones that produce antibodies to the antigen,and isolating the antibodies from the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. For example,humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and then,substituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions.

General techniques for cloning murine immunoglobulin variable domainsare described, for example, by the publication of Orlandi et al., Proc.Natl Acad. Sci. USA 86:3833 (1989), which is incorporated by referencein its entirety. Techniques for constructing chimeric antibodies arewell known to those of skill in the art. As an example, Leung et al.,Hybridoma 13:469 (1994), describe how they produced an LL2 chimera bycombining DNA sequences encoding the V_(κ) and V_(H) domains of LL2monoclonal antibody, an anti-CD22 antibody, with respective human K andIgG₁ constant region domains. This publication also provides thenucleotide sequences of the LL2 light and heavy chain variable regions,V_(K) and V_(H), respectively. Techniques for producing humanized MAbsare described, for example, by Jones et al., Nature 321: 522 (1986),Riechmann et al., Nature 332: 323 (1988), Verhoeyen et al., Science 239:1534 (1988), Carter et al., Proc. Natl Acad. Sci. USA 89: 4285 (1992),Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun.150:2844 (1993), each of which is hereby incorporated by reference.

A chimeric antibody is a recombinant protein that contains the variabledomains including the CDRs derived from one species of animal, such as arodent antibody, while the remainder of the antibody molecule; i.e., theconstant domains, is derived from a human antibody. Accordingly, achimeric monoclonal antibody can also be humanized by replacing thesequences of the murine FR in the variable domains of the chimeric mAbwith one or more different human FR. Specifically, mouse CDRs aretransferred from heavy and light variable chains of the mouseimmunoglobulin into the corresponding variable domains of a humanantibody. As simply transferring mouse CDRs into human FRs often resultsin a reduction or even loss of antibody affinity, additionalmodification might be required in order to restore the original affinityof the murine antibody. This can be accomplished by the replacement ofone or more some human residues in the FR regions with their murinecounterparts to obtain an anatibody that possesses good binding affinityto its epitope. See, for example, Tempest et al., Biotechnology 9:266(1991) and Verhoeyen et al., Science 239: 1534 (1988). Further, theaffinity of humanized, chimeric and human MAbs to a specific epitope canbe increased by mutagenesis of the CDRs, so that a lower dose ofantibody may be as effective as a higher dose of a lower affinity MAbprior to mutagenesis. See for example, WO0029584A1.

Another method for producing the antibodies of the present invention isby production in the milk of transgenic livestock. See, e.g., Colman,A., Biochem. Soc. Symp., 63: 141-147, 1998; U.S. Pat. No. 5,827,690,both of which are incoporated in their entirety by reference. Two DNAconstructs are prepared which contain, respectively, DNA segmentsencoding paired immunoglobulin heavy and light chains. The DNA segmentsare cloned into expression vectors which contain a promoter sequencethat is preferentially expressed in mammary epithelial cells. Examplesinclude, but are not limited to, promoters from rabbit, cow and sheepcasein genes, the cow a-lactoglobulin gene, the sheep β-lactoglobulingene and the mouse whey acid protein gene. Preferably, the insertedfragment is flanked on its 3′ side by cognate genomic sequences from amammary-specific gene. This provides a polyadenylation site andtranscript-stabilizing sequences. The expression cassettes arecoinjected into the pronuclei of fertilized, mammalian eggs, which arethen implanted into the uterus of a recipient female and allowed togestate. After birth, the progeny are screened for the presence of bothtransgenes by Southern analysis. In order for the antibody to bepresent, both heavy and light chain genes must be expressed concurrentlyin the same cell. Milk from transgenic females is analyzed for thepresence and functionality of the antibody or antibody fragment usingstandard immunological methods known in the art. The antibody can bepurified from the milk using standard methods known in the art.

A fully human antibody of the present invention, i.e., human anti-CD20MAbs or other human antibodies, such as anti-CD19, anti-CD22, anti-CD21or anti-CD23 MAbs for combination therapy with humanized, chimeric orhuman anti-CD20 antibodies, can be obtained from a transgenic non-humananimal. See, e.g., Mendez et al., Nature Genetics 15: 146-156 (1997);U.S. Pat. No. 5,633,425, both of which are incorporated in theirentirety by reference. For example, a human antibody can be recoveredfrom a transgenic mouse possessing human immunoglobulin loci. The mousehumoral immune system is humanized by inactivating the endogenousimmunoglobulin genes and introducing human immunoglobulin loci. Thehuman immunoglobulin loci are exceedingly complex and comprise a largenumber of discrete segments which together occupy almost 0.2% of thehuman genome. To ensure that transgenic mice are capable of producingadequate repertoires of antibodies, large portions of human heavy- andlight-chain loci must be introduced into the mouse genome. This isaccomplished in a stepwise process beginning with the formation of yeastartificial chromosomes (YACs) containing either human heavy- orlight-chain immunoglobulin loci in germline configuration. Since eachinsert is approximately 1 Mb in size, YAC construction requireshomologous recombination of overlapping fragments of the immunoglobulinloci. The two YACs, one containing the heavy-chain loci and onecontaining the light-chain loci, are introduced separately into mice viafusion of YAC-containing yeast spheroblasts with mouse embryonic stemcells. Embryonic stem cell clones are then microinjected into mouseblastocysts. Resulting chimeric males are screened for their ability totransmit the YAC through their germline and are bred with mice deficientin murine antibody production. Breeding the two transgenic strains, onecontaining the human heavy-chain loci and the other containing the humanlight-chain loci, creates progeny which produce human antibodies inresponse to immunization.

Further recent methods for producing bispecific mAbs include engineeredrecombinant mAbs which have additional cysteine residues so that theycrosslink more strongly than the more common immunoglobulin isotypes.See, e.g., FitzGerald et al., Protein Eng. 10(10):1221-1225, 1997.Another approach is to engineer recombinant fusion proteins linking twoor more different single-chain antibody or antibody fragment segmentswith the needed dual specificities. See, e.g., Coloma et al., NatureBiotech. 15:159-163, 1997. A variety of bispecific fusion proteins canbe produced using molecular engineering. In one form, the bispecificfusion protein is monovalent, consisting of, for example, a scFv with asingle binding site for one antigen and a Fab fragment with a singlebinding site for a second antigen. In another form, the bispecificfusion protein is divalent, consisting of, for example, an IgG with twobinding sites for one antigen and two scFv with two binding sites for asecond antigen.

Bispecific fusion proteins linking two or more different single-chainantibodies or antibody fragments are produced in similar manner.Recombinant methods can be used to produce a variety of fusion proteins.For example a fusion protein comprising a Fab fragment derived from ahumanized monoclonal anti-CD20 antibody and a scFv derived from a murineanti-diDTPA can be produced. A flexible linker, such as GGGS connectsthe scFv to the constant region of the heavy chain of the anti-CD20antibody. Alternatively, the scFv can be connected to the constantregion of the light chain of another humanized antibody. Appropriatelinker sequences necessary for the in-frame connection of the heavychain Fd to the scFv are introduced into the VL and VK domains throughPCR reactions. The DNA fragment encoding the scFv is then ligated into astaging vector containing a DNA sequence encoding the CHI domain. Theresulting scFv-CH1 construct is excised and ligated into a vectorcontaining a DNA sequence encoding the VH region of an anti-CD20antibody. The resulting vector can be used to transfect an appropriatehost cell, such as a mammalian cell for the expression of the bispecificfusion protein.

Examples of such bivalent and bispecific antibodies are found in U.S.patent application Ser. Nos. 60/399,707, filed Aug. 1, 2002; 60/360,229,filed Mar. 1, 2002; 60/388,314, filed Jun. 14, 2002; and 10/116,116,filed Apr. 5, 2002, all of which are incorporated by reference herein.

3. Production of Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab)₂, Fab′, Fab, Fv, sFv and the like. Otherantibody fragments include, but are not limited to: the F(ab)'₂fragmentswhich can be produced by pepsin digestion of the antibody molecule andthe Fab′ fragments, which can be generated by reducing disulfide bridgesof the F(ab)′₂fragments. Alternatively, Fab′ expression expressionlibraries can be constructed (Huse et al., 1989, Science 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. The present invention encompasses antibodiesand antibody fragments.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). A scFvmolecule is denoted as either VL-L-VH if the VL domain is the N-terminalpart of the scFv molecule, or as VH-L-VL if the VH domain is theN-terminal part of the scFv molecule. Methods for making scFv moleculesand designing suitable peptide linkers are described in U.S. Pat. No.4,704,692, U.S. Pat. No. 4,946,778, R. Raag and M. Whitlow, “SingleChain Fvs.” FASEB 9:73-80 (1995) and R. E. Bird and B. W. Walker, SingleChain Antibody Variable Regions, TIBTECH 9:132-137 (1991). Thesereferences are incorporated herein by reference.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. For example, an antibody fragment can be produced by enzymaticcleavage of antibodies with pepsin to provide a 5S fragment denotedF(ab′)₂. This fragment can be further cleaved using a thiol reducingagent, and optionally a blocking group for the sulfhydryl groupsresulting from cleavage of disulfide linkages, to produce 3.5S Fab′monovalent fragments. Alternatively, an enzymatic cleavage using papainproduces two monovalent Fab fragments and an Fc fragment directly. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89:230 (1960); Porter, Biochem.J. 73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY, Volume 1, p.422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). A CDR is a segment of thevariable region of an antibody that is complementary in structure to theepitope to which the antibody binds and is more variable than the restof the variable region. Accordingly, a CDR is sometimes referred to ashypervariable region. A variable region comprises three CDRs. CDRpeptides can be obtained by constructing genes encoding the CDR of anantibody of interest. Such genes are prepared, for example, by using thepolymerase chain reaction to synthesize the variable region from RNA ofantibody-producing cells. See, for example, Larrick et al., Methods: ACompanion to Methods in Enzymology 2:106 (1991); Courtenay-Luck,“Genetic Manipulation of Monoclonal Antibodies,” in MONOCLONALANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter etal. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds.), pages137-185 (Wiley-Liss, Inc. 1995).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

4. Multispecific and Multivalent Antibodies

The antibodies having the same specificities, as well as other thosehaving different specificities for use in combination therapy, describedherein, can also be made as multispecific antibodies (comprising atleast one binding site to a CD20 epitope or antigen and at least onebinding site to another epitope on CD20 or another antigen) andmultivalent antibodies (comprising mutliple binding sites to the sameepitope or antigen).

The present invention provides a bispecific antibody or antibodyfragment having at least a binding region that specifically binds atargeted cell marker and at least one other binding region thatspecifically binds a targetable conjugate. The targetable conjugatecomprises a carrier portion which comprises or bears at least oneepitope recognized by at least one binding region of the bispecificantibody or antibody fragment.

A variety of recombinant methods can be used to produce bispecificantibodies and antibody fragments as described above.

An multivalent antibody is also contemplated in the present invention.This multivalent target binding protein is constructed by association ofa first and a second polypeptide. The first polypeptide comprises afirst single chain Fv molecule covalently linked to a firstimmunoglobulin-like domain which preferably is an immunoglobulin lightchain variable region domain. The second polypeptide comprises a secondsingle chain Fv molecule covalently linked to a secondimmunoglobulin-like domain which preferably is an immunoglobulin heavychain variable region domain. Each of the first and second single chainFv molecules forms a target binding site, and the first and secondimmunoglobulin-like domains associate to form a third target bindingsite.

A single chain Fv molecule with the VL-L-VH configuration, wherein L isa linker, may associate with another single chain Fv molecule with theVH-L-VL configuration to form a bivalent dimer. In this case, the VLdomain of the first scFv and the VH domain of the second scFv moleculeassociate to form one target binding site, while the VH domain of thefirst scFv and the VL domain of the second scFv associate to form theother target binding site.

Another embodiment of the present invention is bispecific, trivalenttargeting protein comprising two heterologous polypeptide chainsassociated non-covalently to form three binding sites, two of which haveaffinity for one target and a third which has affinity for a hapten thatcan be made and attached to a carrier for a diagnostic and/ortherapeutic agent. Preferably, the binding protein has two similarantigenic binding sites and a different antigenic binding site. Thebispecific, trivalent targeting agents have two different scFvs, onescFv contains two V_(H) domains from one antibody connected by a shortlinker to the V_(L) domain of another antibody and the second scFvcontains two V_(L) domains from the first antibody connected by a shortlinker to the V_(H) domain of the other antibody. The methods forgenerating multivalent, multispecific agents from V_(H) and V_(L)domains provide that individual chains synthesized from a DNA plasmid ina host organism are composed entirely of V_(H) domains (the V_(H)-chain)or entirely of V_(L) domains (the V_(L)-chain) in such a way that anyagent of multivalency and multispecificity can be produced bynon-covalent association of one V_(H)-chain with one V_(L)-chain. Forexample, forming a trivalent, trispecific agent, the V_(H)-chain willconsist of the amino acid sequences of three V_(H) domains, each from anantibody of different specificity, joined by peptide linkers of variablelengths, and the V_(L)-chain will consist of complementary V_(L)domains, joined by peptide linkers similar to those used for theV_(H)-chain. Since the V_(H) and V_(L) domains of antibodies associatein an anti-parallel fashion, the preferred method in this invention hasthe V_(L) domains in the V_(L)-chain arranged in the reverse order ofthe V_(H) domains in the V_(H)-chain.

5.Diabodies, Triabodies and Tetrabodies

The antibodies of the present invention can also be used to preparefunctional bispecific single-chain antibodies (bscAb), also calleddiabodies, and can be produced in mammalian cells using recombinantmethods. See, e.g., Mack et al., Proc. Natl. Acad. Sci., 92:7021-7025,1995, incorporated. For example, bscAb are produced by joining twosingle-chain Fv fragments via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest are isolated using standard PCR methods. TheV_(L) and V_(H) cDNA's obtained from each hybridoma are then joined toform a single-chain fragment in a two-step fusion PCR. The first PCRstep introduces the (Gly₄-Ser₁)₃ linker, and the second step joins theV_(L) and V_(H) amplicons. Each single chain molecule is then clonedinto a bacterial expression vector. Following amplification, one of thesingle-chain molecules is excised and sub-cloned into the other vector,containing the second single-chain molecule of interest. The resultingbscAb fragment is subcloned into an eukaryotic expression vector.Functional protein expression can be obtained by transfecting the vectorinto chinese hamster ovary cells. Bispecific fusion proteins areprepared in a similar manner. Bispecific single-chain antibodies andbispecific fusion proteins are included within the scope of the presentinvention.

For example, a humanized, chimeric or human anti-CD22 monoclonalantibody can be used to produce antigen specific diabodies, triabodies,and tetrabodies. The monospecific diabodies, triabodies, and tetrabodiesbind selectively to targeted antigens and as the number of binding siteson the molecule increases, the affinity for the target cell increasesand a longer residence time is observed at the desired location. Fordiabodies, the two chains comprising the V_(H) polypeptide of thehumanized CD22 MAb connected to the V_(K) polypeptide of the humanizedCD22 MAb by a five amino acid residue linker are utilized. Each chainforms one half of the humanized CD22 diabody. In the case of triabodies,the three chains comprising V_(H) polypeptide of the humanized CD22 MAbconnected to the V_(K) polypeptide of the humanized CD22 MAb by nolinker are utilized. Each chain forms one third of the hCD22 triabody.

The preferred use of the bispecific diabodies described herein is forpre-targeting CD22 positive tumors for subsequent specific delivery ofdiagnostic or therapeutic agents. These diabodies bind selectively totargeted antigens allowing for increased affinity and a longer residencetime at the desired location. Moreover, non-antigen bound diabodies arecleared from the body quickly and exposure of normal tissues isminimized The diagnostic and therapeutic agents can include isotopes,drugs, toxins, cytokines, hormones, growth factors, conjugates,radionuclides, and metals. For example, gadolinium metal is used formagnetic resonance imaging (MRI). Examples of radionuclides are ²²⁵Ac,¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In, ¹³¹I, ^(99m)Tc, ^(94m) _(Tc), ¹⁸⁶Re,¹⁸⁸Re, ¹⁷⁷Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br,and ²¹¹At. Other radionuclides are also available as diagnostic andtherapeutic agents, especially those in the energy range of 60 to 4,000keV.

More recently, a tetravalent tandem diabody (termed tandab) with dualspecificity has also been reported (Cochlovius et al., Cancer Research(2000) 60:4336-4341). The bispecific tandab is a dimer of two identicalpolypeptides, each containing four variable domains of two differentantibodies (V_(H1), V_(L1), V_(H2), V_(L2)) linked in an orientation tofacilitate the formation of two potential binding sites for each of thetwo different specificities upon self-association.

6. Conjugated multivalent and Multispecific Antibodies

In another embodiment of the instant invention is a conjugatedmultivalent antibody. Additional amino acid residues may be added toeither the N- or C-terminus of the first or the second polypeptide. Theadditional amino acid residues may comprise a peptide tag, a signalpeptide, a cytokine, an enzyme (for example, a pro-drug activatingenzyme), a hormone, a peptide toxin, such as pseudomonas extoxin, apeptide drug, a cytotoxic protein or other functional proteins. As usedherein, a functional protein is a protein which has a biologicalfunction.

In one embodiment, drugs, toxins, radioactive compounds, enzymes,hormones, cytotoxic proteins, chelates, cytokines and other functionalagents may be conjugated to the multivalent target binding protein,preferably through covalent attachments to the side chains of the aminoacid residues of the multivalent target binding protein, for exampleamine, carboxyl, phenyl, thiol or hydroxyl groups. Various conventionallinkers may be used for this purpose, for example, diisocyanates,diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,maleimide-hydroxysuccinimide esters, glutaraldehyde and the like.Conjugation of agents to the multivalent protein preferably does notsignificantly affect the protein's binding specificity or affinity toits target. As used herein, a functional agent is an agent which has abiological function. A preferred functional agent is a cytotoxic agent.

In still other embodiments, bispecific antibody-directed delivery oftherapeutics or prodrug polymers to in vivo targets can be combined withbispecific antibody delivery of radionuclides, such that combinationchemotherapy and radioimmunotherapy is achieved. Each therapy can beconjugated to the targetable conjugate and administered simultaneously,or the nuclide can be given as part of a first targetable conjugate andthe drug given in a later step as part of a second targetable conjugate.

In another embodiment, cytotoxic agents may be conjugated to a polymericcarrier, and the polymeric carrier may subsequently be conjugated to themultivalent target binding protein. For this method, see Ryser et al .,Proc. Natl. Acad. Sci. USA, 75:3867-3870 (1978), U.S. Pat. No. 4,699,784and U.S. Pat. No. 4,046,722, which are incorporated herein by reference.Conjugation preferably does not significantly affect the bindingspecificity or affinity of the multivalent binding protein.

7. Use of Subhuman Primatized, Humanized, Chimeric and Human Antibodiesfor Treatment and Diagnosis

Subhuman primatized, humanized, chimeric and human monoclonalantibodies, i.e., anti-CD20 MAbs and other MAbs described herein, inaccordance with this invention are suitable for use in therapeuticmethods and diagnostic methods. Accordingly, the present inventioncontemplates the administration of the subhuman primatized, humanized,chimeric and human antibodies of the present invention alone as a nakedantibody or administered as a multimodal therapy, temporally accordingto a dosing regimen, but not conjugated to, a therapeutic agent. Theefficacy of the naked anti-CD20 MAbs can be enhanced by supplementingnaked antibodies with one or more other naked antibodies, i.e., MAbs tospecific antigens, such as CD4, CD5, CD8, CD14, CD15, CD19, CD21, CD22,CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD46, CD52, CD54, CD74, CD80,CD126, B7, Ia, HM1.24, tenascin, MUC1, or HLA-DR, as well as withantiangiogenesis antibodies (e.g., VEGF and P1GF antibodies) with one ormore immunoconjugates of anti-CD20, or antibodies to theses recitedantigens, conjugated with therapeutic agents, including drugs, toxins,immunomodulators, hormones, therapeutic radionuclides, etc., with one ormore therapeutic agents, including drugs, toxins, immunomodulators,hormones, therapeutic radionuclides, etc., administered concurrently orsequentially or according to a prescribed dosing regimen, with the MAbs.Preferred B-cell antigens include those equivalent to human CD19, CD20,CD21, CD22, CD23, CD46, CD52, CD74, CD80, and CD5 antigens. PreferredT-cell antigens include those equivalent to human CD4, CD8 and CD25 (theIL-2 receptor) antigens. An equivalent to HLA-DR antigen can be used intreatment of both B-cell and T-cell disorders. Particularly preferredB-cell antigens are those equivalent to human CD19, CD22, CD21, CD23,CD74, CD80, and HLA-DR antigens. Particularly preferred T-cell antigensare those equivalent to human CD4, CD8 and CD25 antigens. CD46 is anantigen on the surface of cancer cells that block complement-dependentlysis (CDC).

Further, the present invention contemplates the administration of animmunoconjugate for therapeutic uses in B cell lymphomas and otherdisease or disorders. An immunoconjugate, as described herein, is amolecule comprising an antibody component and a therapeutic agent,including a peptide which may bear the or therapeutic agent. Animmunoconjugate retains the immunoreactivity of the antibody component,i.e., the antibody moiety has about the same or slightly reduced abilityto bind the cognate antigen after conjugation as before conjugation.

Also, the present invention contemplates the administration of animmunoconjugate for therapeutic uses in myeloid leukemias, in whichCD33, CD45, CD66, and other granulocyte-associated antigens aretargeted.

A wide variety of therapeutic reagents can be advantageously conjugatedto the antibodies of the invention. The therapeutic agents recited hereare those agents that also are usefulfor administration separately withthe naked antibody as described above. Therapeutic agents include, forexample, chemotherapeutic drugs such as vinca alkaloids, anthracyclines,epidophyllotoxins, taxanes, antimetabolites, alkylating agents,antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic andapoptotoic agents, particularly doxorubicin, methotrexate, taxol,CPT-11, camptothecans, and others from these and other classes ofanticancer agents , and the like. Other useful cancer chemotherapeuticdrugs for the preparation of immunoconjugates and antibody fusionproteins include nitrogen mustards, alkyl sulfonates, nitrosoureas,triazenes, folic acid analogs, COX-2 inhibitors, pyrimidine analogs,purine analogs, platinum coordination complexes, including oxaliplatin,hormones, and the like. Suitable chemotherapeutic agents are describedin REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co.,1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OFTHERAPEUTICS, 7th Ed. (MacMillan Publishing Co., 1985), as well asrevised editions of these publications. Other suitable chemotherapeuticagents, such as experimental drugs, are known to those of skill in theart.

Additionally, a chelator such as DTPA, DOTA, TETA, or NOTA or a suitablepeptide, to which a detectable label, such as a fluorescent molecule, orcytotoxic agent, such as a heavy metal or radionuclide, can beconjugated. For example, a therapeutically useful immunoconjugate can beobtained by conjugating a photoactive agent or dye to an antibodycomposite. Fluorescent compositions, such as fluorochrome, and otherchromogens, or dyes, such as porphyrins sensitive to visible light, havebeen used to detect and to treat lesions by directing the suitable lightto the lesion. In therapy, this has been termed photoradiation,phototherapy, or photodynamic therapy (Joni et al. (eds.), PHOTODYNAMICTHERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van denBergh, Chem. Britain 22:430 (1986)). Moreover, monoclonal antibodieshave been coupled with photoactivated dyes for achieving phototherapy.Mew et al., J. Immunol. 130:1473 (1983); idem., Cancer Res. 45:4380(1985); Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986);idem., Photochem. Photobiol. 46:83 (1987); Hasan et al., Prog. Clin.Biol. Res. 288:471 (1989); Tatsuta et al., Lasers Surg. Med. 9:422(1989); Pelegrin et al., Cancer 67:2529 (1991). However, these earlierstudies did not include use of endoscopic therapy applications,especially with the use of antibody fragments or subfragments. Thus, thepresent invention contemplates the therapeutic use of immunoconjugatescomprising photoactive agents or dyes.

A toxin, such as Pseudomonas exotoxin, may also be complexed to or formthe therapeutic agent portion of an antibody fusion protein of ananti-CD20 antibody of the present invention. Other toxins suitablyemployed in the preparation of such conjugates or other fusion proteins,include ricin, abrin, ribonuclease (RNase), DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, Calif.—A CancerJournal for Clinicians 44:43 (1994). Additional toxins suitable for usein the present invention are known to those of skill in the art and aredisclosed in U.S. Pat. No. 6,077,499, which is incorporated in itsentirety by reference.

An immunomodulator, such as a cytokine may also be conjugated to, orform the therapeutic agent portion of an antibody fusion protein or beadministered with the humanized anti-CD20 or other lymphoma antibodiesof the present invention. Suitable cytokines for the present inventioninclude, but are not limited to, interferons and interleukins, asdescribed below.

8.Preparation of Immunoconjugates

Any of the antibodies or antibody fusion proteins of the presentinvention can be conjugated with one or more therapeutic agent.Generally, one therapeutic agent is attached to each antibody orantibody fragment, but more than one therapeutic agent agent can beattached to the same antibody or antibody fragment. The antibody fusionproteins of the present invention comprise two or more antibodies orfragments thereof and each of the antibodies that composes this fusionprotein can contain a therapeutic agent agent. Additionally, one or moreof the antibodies of the antibody fusion protein can have more than onetherapeutic agent attached. Further, the therapeutic agents do not needto be the same but can be different therapeutic agents. For example, onecan attach a drug and a radioisotope to the same fusion protein.Particulary, an IgG can be radiolabeled with ¹³¹I and attached to adrug. The ¹³¹I can be incorporated into the tyrosine of the IgG and thedrug attached to the epsilon amino group of the IgG lysines. Thetherapeutic agents also can be attached to reduced SH groups and to thecarbohydrate side chains.

Bispecific antibodies of the present invention are useful inpretargeting methods and provide a preferred way to deliver twotherapeutic agents to a subject. U.S. Ser. No. 09/382,186 discloses amethod of pretargeting using a bispecific antibody, in which thebispecific antibody is labeled with ¹²⁵I and delivered to a subject,followed by a divalent peptide labeled with ^(99m)Tc. The deliveryresults in excellent tumor/normal tissue ratios for ¹²⁵I and ^(99m)Tc,thus showing the utility of two diagnostic radioisotopes. Anycombination of known therapeutic agents agents can be used to label theantibodies and antibody fusion proteins. The binding specificity of theantibody component of the mAb conjugate, the efficacy of the therapeuticagent or diagnostic agent and the effector activity of the Fc portion ofthe antibody can be determined by standard testing of the conjugates.

A therapeutic agent can be attached at the hinge region of a reducedantibody component via disulfide bond formation. As an alternative, suchpeptides can be attached to the antibody component using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56: 244(1994). General techniques for such conjugation are well-known in theart. See, e.g., Wong, CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING(CRC Press, 1991); Upeslacis et al., “Modification of Antibodies byChemical Methods,” in MONOCLONAL ANTIBODIES: PRINCIPLES ANDAPPLICATIONS, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.,1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES: PRODUCTION,ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 60-84(Cambridge University Press, 1995). Alternatively, the therapeutic agentcan be conjugated via a carbohydrate moiety in the Fc region of theantibody. The carbohydrate group can be used to increase the loading ofthe same peptide that is bound to a thiol group, or the carbohydratemoiety can be used to bind a different peptide.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, all of which are incorporated in their entirety by reference.The general method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function and that is loaded with a plurality of peptide.This reaction results in an initial Schiff base (imine) linkage, whichcan be stabilized by reduction to a secondary amine to form the finalconjugate.

The Fc region is absent if the antibody used as the antibody componentof the immunoconjugate is an antibody fragment. However, it is possibleto introduce a carbohydrate moiety into the light chain variable regionof a full length antibody or antibody fragment. See, for example, Leunget al., J. Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No.5,443,953 (1995), Leung et al., U.S. Pat. No. 6,254,868, all of whichare incoporated in their entirety by reference. The engineeredcarbohydrate moiety is used to attach the therapeutic or diagnosticagent.

9. Pharmaceutically Acceptable Vehicles

The subhuman primatized, humanized, chimeric or human radiolabeledantibody to be delivered to a subject can consist of the mAb alone,immunoconjugate, fusion protein, or can comprise one or morepharmaceutically suitable vehicles, one or more additional ingredients,or some combination of these.

The immunoconjugate antibody of the present invention can be formulatedaccording to known methods to prepare pharmaceutically usefulcompositions, whereby the immunoconjugate or naked antibody is combinedin a mixture with a pharmaceutically suitable vehicle. Sterilephosphate-buffered saline is one example of a pharmaceutically suitablevehicle. Other suitable vehicles are well-known to those in the art.See, for example, Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUGDELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

The immunoconjugate or naked antibody of the present invention can beformulated for parenteral application, such as intravenousadministration via, for example, bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the therapeutic conjugate or naked antibody.Control-release preparations can be prepared through the use of polymersto complex or adsorb the immunoconjugate or naked antibody. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992).The rate of release of an immunoconjugate or antibody from such a matrixdepends upon the molecular weight of the immunoconjugate or antibody,the amount of immunoconjugate, antibody within the matrix, and the sizeof dispersed particles. Saltzman et al., Biophys. J. 55: 163 (1989);Sherwood et al., supra. Other solid dosage forms are described in Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The immunoconjugate, antibody fusion proteins, or naked antibody mayalso be administered to a mammal subcutaneously or even by otherparenteral routes. Moreover, the administration may be by continuousinfusion or by single or multiple boluses. In general, the dosage of anadministered immunoconjugate, fusion protein or naked antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, it is desirable to provide the recipient with adosage of immunoconjugate, antibody fusion protein or naked antibodythat is in the range of from about 1 mg/kg to 20 mg/kg as a singleintravenous infusion, although a lower or higher dosage also may beadministered as circumstances dictate. This dosage may be repeated asneeded, for example, once per week for 4-10 weeks, preferably once perweek for 8 weeks, and more preferably, once per week for 4 weeks. It mayalso be given less frequently, such as every other week for severalmonths. The dosage may be given through various parenteral routes, withappropriate adjustment of the dose and schedule.

For purposes of therapy, the immunoconjugate, fusion protein, or nakedantibody is administered to a mammal in a therapeutically effectiveamount. A suitable subject for the present invention is usually a human,although a non-human animal subject is also contemplated. An antibodypreparation is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient mammal. Inparticular, an antibody preparation of the present invention isphysiologically significant if its presence invokes an antitumorresponse or mitigates the signs and symptoms of an autoimmune diseasestate. A physiologically significant effect could also be the evocationof a humoral and/or cellular immune response in the recipient mammal

10. Methods of Treatment

The present invention contemplates the use antibodies of the presentinvention as the primary composition for treatment of diseases such as aB-cell related malignancy, a T-cell malignancy or another lymphoma type.In addition, it is also useful for treating autoimmune diseases. Inparticular, the compositions described herein are particularly usefulfor treatment of various autoimmune diseases as well as indolent formsof B-cell lymphomas, aggressive forms of B-cell lymphomas, chroniclymphatic leukemias, acute lymphatic leukemias, and Waldenstrom'smacroglobulinemia, multiple myeloma. Also, T-cell diseases such asT-cell leukemia or mycosis fungoides can be treated. For example, thehumanized anti-CD22 antibody components and immunoconjugates can be usedto treat both indolent and aggressive forms of non-Hodgkin's lymphoma.An autoimmune disease is selected from the group consisting of acuteidiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenicpurpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemiclupus erythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis,sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy,polyarteritis nodosa, ankylosing spondylitis, Goodpasture's syndrome,thromboangitis ubiterans, Sjogren's syndrome, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic activehepatitis, polymyositis/dermatomyositis, polychondritis, pamphigusvulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophiclateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia,pernicious anemia, rapidly progressive glomerulonephritis, psoriasis,and fibrosing alveolitis.

The compositions for treatment contain at least one humanized, chimericor human monoclonal antibody alone or in combination with otherantibodies, such as other humanized, chimeric, or human antibodies,therapeutic agents or immunomodulators. In particular, combinationtherapy with a fully human antibody is also contemplated and is producedby the methods as set forth above.

Conjugated antibodies to the same or different epitope or antigen may bealso be combined with one or more of the antibodies of the presentinvention. For example, a humanized, chimeric or human conjugatedanti-CD22 antibody may be combined with another subhuman primatized,humanized, chimeric or human conjugated anti-CD22, a subhumanprimatized, humanized, chimeric or human conjugated anti-CD22 antibodymay be combined with an anti-CD22 immunoconjugate. Alternatively,various such combinataions can be made with differentlymphoma-associated antibodies, as described above. A fusion protein ofa subhuman primatized, humanized, chimeric or human CD22 antibody and atoxin or immunomodulator, or a fusion protein of at least two differentB-cell antibodies (e.g., a CD20 and a CD22 mAb) may also be used in thisinvention. Many different antibody combinations, targeting at least twodifferent antigens associated with B-cell or other lymphoma orautoimmune disorders, as listed already above, may be constructed,either as partly conjugated with a therapeutic agent or immunomodulator,or merely in combination with another therapeutic agents, such as acytotoxic drug or with radiation.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, such as tumor necrosis factor (TNF), andhematopoietic factors, such as interleukins (e.g., interleukin-1 (IL-1),IL-2, IL-3, IL-6, IL-10, IL-12 and IL-18), colony stimulating factors(e.g., granulocyte-colony stimulating factor (G-CSF) and granulocytemacrophage-colony stimulating factor (GM-CSF)), interferons (e.g.,interferons-α, -β and -γ), the stem cell growth factor designated “Sifactor,” erythropoietin and thrombopoietin. Examples of suitableimmunomodulator moieties include IL-2, IL-6, IL-10, IL-12, IL-18,interferon-γ, TNF-α, and the like. Alternatively, subjects can receiveconjugated anti-CD20 antibodies and a separately administered cytokine,which can be administered before, concurrently or after administrationof the naked or conjugated anti-CD20 antibodies. As discussed supra, theanti-CD22 antibody may also be conjugated to the immunomodulator. Theimmunomodulator may also be conjugated to a hybrid antibody or hybridantibody fragments or subfragments (single-chain binding proteins, orsFv's) consisting of one or more antibodies or subfragments binding todifferent antigens.

Multimodal therapies of the present invention further includeimmunotherapy with conjugated anti-CD22 antibodies supplemented withadministration of anti-CD20, anti-CD19, anti-CD21, anti-CD74, anti-CD80,anti-CD23, anti-CD46 or HLA-DR (including the invariant chain)antibodies in the form of fusion proteins or as immunoconjugates. Theseantibodies include polyclonal, monoclonal, primatized subhuman,chimeric, human or humanized antibodies that recognize at least oneepitope on these antigenic determinants. Anti-CD19 and anti-CD22antibodies are known to those of skill in the art. See, for example,Ghetie et al., Cancer Res. 48:2610 (1988); Hekman et al., CancerImmunol. Immunother. 32:364 (1991); Longo, Curr. Opin. Oncol. 8:353(1996) and U.S. Pat. Nos. 5,798,554 and 6,187,287, incorporated in theirentirety by reference.

In another form of multimodal therapy, subjects receive conjugatedantibodies, and/or immunoconjugates, in conjunction with standard cancerchemotherapy. For example, “CVB” (1.5 g/m² cyclophosphamide, 200-400mg/m² etoposide, and 150-200 mg/m² carmustine) is a regimen used totreat non-Hodgkin's lymphoma. Patti et al., Eur. J. Haematol. 51: 18(1993). Other suitable combination chemotherapeutic regimens arewellknown to those of skill in the art. See, for example, Freedman etal., “Non-Hodgkin's Lymphomas,” in CANCER MEDICINE, VOLUME 2, 3rdEdition, Holland et al. (eds.), pages 2028-2068 (Lea & Febiger 1993). Asan illustration, first generation chemotherapeutic regimens fortreatment of intermediate-grade non-Hodgkin's lymphoma (NHL) includeC-MOPP (cyclophosphamide, vincristine, procarbazine and prednisone) andCHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Auseful second-generation chemotherapeutic regimen is m-BACOD(methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine,dexamethasone and leucovorin), while a suitable third generation regimenis MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine,prednisone, bleomycin and leucovorin). Additional useful drugs includephenyl butyrate and brostatin-1. In a preferred multimodal therapy, bothchemotherapeutic drugs and cytokines are co-administered with anantibody, immunoconjugate or fusion protein according to the presentinvention. The cytokines, chemotherapeutic drugs and antibody orimmunoconjugate can be administered in any order, or together.

Radionuclides useful as therapeutic agents, which substantially decay bybeta-particle emission include, include but are not limited to Ac-225,P-32, P-33, Sc-47, Fe-59, Cu-64, Cu-67, Se-75, As-77, Sr-89, Y-90,Mo-99, Rh-105, Pd-109, Ag-111, 1-125, I-131, Pr-142, Pr-143, Pm-149,Sm-153, Tb-161, Ho-166, Er-169, Lu-177, Re-186, Re-188, Re-189, Ir-194,Au-198, Au-199, Pb-211, Pb-212, and Bi-213. Maximum decay energies ofuseful beta-particle-emitting nuclides are preferably 20-5,000 keV, morepreferably 100-4,000 keV, and most preferably 500-2,500 keV.

Radionuclides useful as therapeutic agents, which substantially decaywith Auger-emitting particles include, but are not limited to Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, I-125, Ho-161,Os-189m and Ir-192. Maximum decay energy of these radionuclides ispreferably less than 1,000 keV, more preferably less than 100 keV, andmost preferably less than 70 keV.

Radionuclides useful as therapeutic agents, which substantially decaywith generation of alpha-particles include, but are not limited toDy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221,At-217, Bi-213 and Fm-255. Decay energies of usefulalpha-particle-emitting radionuclides are preferably 2,000-9,000 keV,more preferably 3,000-8,000 keV, and most preferably 4,000-7,000 keV.

Radionuclides useful in therapies based on neutron capture proceduresinclude, but are not limited to B-10, Gd-157 and U-235.

The embodiments of the invention may be further illustrated throughexamples which show aspects of the invention in detail. These examplesillustrate specific elements of the invention and are not to beconstrued as limiting the scope thereof.

Examples Antibodies

Epratuzumab is a humanized LL2 antibody and developed by ImmunomedicsInc., Morris Plains, N.J.. The humanization process replaces ˜95% of themurine Ig sequence with the human IgG₁ sequence. Epratuzumab isinternalized once it binds to the B epitope of the CD22 antigen (Stein,R. et al., Cancer Immunol. Immunother. 37(5):293-8, 1993) thatcorresponds to the third Ig domain (Kehrl, J. H. B6 CD22 Workshop PanelReport, in Leukocyte Typing V. White Cell Differentiation Antigens., S.F. Schlossman (ed.), Oxford University Press, p. 523-5, 1995). In vitrointernalization was observed after five minutes and re-expression of asmuch as 50% of the antigen was reported to occur after 5 hours (Shih, L.B. et al. Int J Cancer 56(4):538-45, 1994).

The humanized anti-CD20 antibody, hA20, has been developed byImmunomedics, Inc., Morris Plains, N.J.. This Mab binds to CD20 and, incontrast to the chimeric anti-CD20 MAb, rituximab, it is a CDR-graftedMAb that has less murine protein than the chimeric form. The hA20 MAbhas IgG1(kappa) constant regions and the same humanV framework regionasas epratuzumab, the CD22 humanized MAb. The genes of CDR-grafted VH andVk chains of hA20 were inserted into the pdHL2 plasmid vector, aDHFR-based amplifiable expression system, and transfected into the Sp2/0murine myeloma cell line to generate the hA20-producing clones.Molecular characterization demonstrated that hA20 is similar, in itsCDRs, to rituximab, except for one amino acid difference in the VHregion. However, differences in the VH and Vk framework regions of hA20,due to the inclusion of more human constructs, are present. This hA20antibody appears to compete with the binding of rituximab for variouslymphoma cells, and has a similar dissociation constant to rituximab, aswell as similar effects in vitro and in vivo against human lymphoma celllines expressing CD20.

Therapy of Non-Hodgkin's Lymphoma (NHL)

A 66-year-old man presents with stage IV diffuse-large cell NHL, havingrelapsed after 3 courses of chemotherapy given in the prior two years.He is given a dose of two injections of ⁹⁰Y-DOTA-epratuzumab (aslabelled in accordance with Govinden, ibid.), one week apart, having 7.5mCi/m² of ⁹⁰Y administered by intravenous infusion with a total dose of30 mg antibody protein each time. Six weeks later, his cervical lymphnodes and his splenomegaly appear to have been reduced markedly, and thepatient is symptomatically improved and returns to work full time. Sincehe does not have a complete remission, a continuous therapy isinstituted involving a combination of epratuzumab (360 mg/m² and hA20(250 mg/m2), given every other week for a total of 4 infusions, and thenthe combined antibody therapy course is repeated 12 weeks later. Threemonths after completion of the second therapy course with thecombination of naked CD22 and CD20 antibodies, the patient has noevidence of disease by radiological scans or bone marrow biopsy, and isthus considered to be a complete response. At the next evaluation, 3months later, his is still in a complete remission of his disease.

Therapy of T-Cell Leukemia

A patient refractive to prior chemotherapy and with advanced T-cellleukemia is given an infusion of 50 mg anti-CD25 humanized Mabconjugated with 20 mCi ⁹⁰Y-DOTA, followed one week later with aninfusion of CD25 Mab (anti-TAC humanized antibody) at a dose of 200mg/m². Four weeks later, his blood count and marrow biopsy indicate apartial remission of his disease.

Therapy of Refractive Rheumatoid Arthritis

A patient presenting with severe, advanced rheumatoid arthritisaffecting many joints, but particularly his knees, and now refractive tochemotherapy, is treated with a single infusion of a mixture of CD4 andCD20 humanized Mabs, totalling 50 mg, labelled with ⁹⁰Y at a dose of 10mCi/m². Two weeks later, he is given a dose of naked humanizedantibodies consisting of 100 mg CD4 and 250 mg CD20 antibodies, and thisis repeated once again two weeks later. The patient feels relief of hisarthritis, particularly in his knees, 4 weeks later, and is able to walkbetter and even climb stairs, with almost no joint inflammation noted byhis physician. Three months later, this course of therapy involving oneinfusion of the radiolabelled mixture of antibodies, followed by twoinfusions of naked CD4 and CD20 antibodies, is repeated, and the patientre-evaluated 6 weeks later. The physician notes marked improvement, suchthat the patient evidences only minimal pain and considerably bettermobility of his extremities.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention, which isdefined by the following embodiments.

All of the publications and patent applications and patents cited inthis specification are herein incorporated in their entirety byreference.

1. A method for treating a disease in a mammal comprising administeringto said mammal concurrently or sequentially a therapeutic compositioncomprising a pharmaceutically acceptable vehicle and at least oneconjugated antibody or a fragment thereof or a conjugated antibodyfusion protein or a fragment thereof, wherein predosing with anon-radiolabeled antibody is not performed, wherein said disease is a Bcell-related disease, a T-cell related disease or an autoimmune disease.2. The method of claim 1, wherein an unconjugated antibody or fragmentor unconjugated antibody fusion protein or fragment thereof isoptionally added with said conjugated antibody or said fragment thereofor said conjugated antibody fusion protein or said fragment thereof, asa maintenance therapy to keep proliferating tumor or autoimmune-diseasedcells from target escape.
 3. The method of claim 2, wherein saidconjugated and unconjugated antibody, antibody fusion protein, orfragment thereof is targeted against an antigen selected from the groupconsisting of CD3, CD4, CD5, CD8, CD11c, CD14, CD15, CD19, CD20, CD21,CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD4OL, CD46, CD52, CD54, CD74,CD80, CD126, MUC1, tenascin, Ia, HMI.24, HLA-DR, and a tumor-associatedantigen. 4-5. (canceled)
 6. The method of claim 3, wherein saidconjugated and unconjugated antibody, antibody fusion protein, orfragment thereof is human, murine, chimeric, primatized or humanized. 7.The method of claim 3, wherein said antibody, antibody fusion protein,or fragment thereof is conjugated to a therapeutic agent selected fromthe group consisting of drug, toxin, immunomodulator, chelator, boroncompounds, photoactive agent, and radionuclide.
 8. The method of claim3, further comprising administering to said mammal concurrently orsequentially a therapeutic composition comprising a pharmaceuticallyacceptable vehicle and at least one antibody, antibody fusion protein orfragment thereof, wherein said antibody, said antibody fusion protein orsaid fragment thereof is conjugated to at least one therapeutic agent.9-14. (canceled)
 15. The method of claim 1, further comprisingadministering to said mammal concurrently or sequentially a therapeuticcomposition comprising a pharmaceutically acceptable vehicle and anantibody fusion protein or fragment thereof that comprises at least twoantibodies or fragments thereof.
 16. The method of claim 15, wherein anunconjugated antibody is optionally added with said antibody fusionprotein or fragment thereof, as a maintenance therapy to keepproliferating tumor or other diseased cells from target escape.
 17. Themethod of claims 16, wherein said conjugated and unconjugated antibodyis targeted against an antigen selected from the group consisting ofCD3, CD4, CD5, CD8, CD11c, CD14, CD15, CD19, CD20, CD21, CD22, CD23,CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD74, CD80,CD126, MUC1, tenascin, Ia, HMI.24, HLA-DR, and a tumor-associatedantigen. 18-19. (canceled)
 20. The method of claim 17, wherein saidconjugated and unconjugated antibody is human, murine, chimeric,primatized or humanized.
 21. The method of claim 17, wherein saidantibody is conjugated to a therapeutic agent selected from the groupconsisting of drug, toxin, immunomodulator, chelator, boron compounds,photoactive agent, and radionuclide. 22-47. (canceled)
 48. The method ofclaim 1, wherein said B-cell related disease is an indolent form ofB-cell lymphoma, an aggressive form of B-cell lymphoma, a chroniclymphocytic leukemia, an acute lymphocytic leukemia, a Waldenström'smacroglobulinemia, or a multiple myeloma.
 49. (canceled)
 50. The methodof claim 1, wherein said B cell lymphoma is a non-Hodgkin's lymphoma.51. The method of claim 1, wherein said T-cell related disease is ahuman or veterinary T-cell leukemia or mycosis fungoides. 52-54.(canceled)
 55. The method of claim 1, wherein said conjugated andunconjugated antibodies, antibody fusion proteins or fragments thereofare directed to the same or different targets.
 56. The method of claim1, wherein said conjugated and unconjugated antibodies, antibody fusionproteins, or fragments thereof are selected from the group consisting ofintact IgG, F(ab′)₂, F(ab)₂, Fab′, Fab, scFvs, diabodies, triabodies ortetrabodies.
 57. The method of claim 1, wherein said conjugated andunconjugated antibody is an anti-CD22 monoclonal antibody.
 58. Themethod of claim 1, wherein said conjugated and unconjugated antibody isa humanized LL2 monoclonal antibody.
 59. (canceled)
 60. The method ofclaim 57, wherein said anti-CD22 monoclonal antibody is human. 61-65.(canceled)
 66. A method for treating a disease in a mammal comprisingadministering to said mammal a therapeutic composition comprising apharmaceutically acceptable vehicle and a multispecific multivalentantibody, fragment or fusion protein conjugate that binds to at leastone target antigen and a therapeutic agent, wherein predosing with anon-radiolabeled antibody is not performed, wherein said disease is a Bcell-related disease, a T-cell related disease or an autoimmune disease.67. A method for treating a disease in mammals, comprising (a)administering to said mammal a composition that comprises amultispecific multivalent antibody, fragment or fusion protein thatbinds to at least one target antigen; (B) optionally, a clearing agentto allow the composition to clear non-localized antibodies fromcirculation; and (C) administering to said mammal a pharmaceutiallyeffective amount of therapeutic conjugate that binds to saidmultispecific multivalent antibody, fragment or fusion protein, andwherein predosing with a non-radiolabeled antibody is not performed and,wherein said disease is a B cell-related disease, a T-cell relateddisease or an autoimmune disease.